^aP<0.05, vs S4; ^cP<0.05, S3.

Table 3  Correlation between serum fibrotic markers and inflammatory grades

^aP<0.05, vs S4; ^cP<0.05, S3.

Changes of liver fibrotic stages and inflammatory grades
Based on the liver fibrotic stages and inflammatory grades before and after treatment, it was rated as improved (alleviated by ≥1 stage), stable, or deteriorated (≥1 stage). The improvement rates of inflammatory grades and fibrotic stages were 40.0 % and 36.67 % respectively in SA-B group, which were slighting higher than in IFN-g group (36.67 % and 30.0 %). Deterioration rates were 16.7 % and 20.0 % in SA-B group, which were much lower than in IFN-g group (20.0 % and 30.0 %). There was no significant difference between the two groups. Fibrotic stages of 4 patients in SA-B group decreased by 2 stages after treatment. But there was none in IFN-g group.

Changes of serum fibrotic markers
There was no significant differences in serum HA, LM and P-III-P contents between the two groups before treatment. But the serum IV-C content in SA-B group was obviously higher than that in IFN-g group before treatment. The average content of serum HA in SA-B group decreased remarkably after treatment (P<0.05), and decreased in IFN-g group, but with no significant difference. The abnormal rate of serum HA content in SA-B decreased from 80 % to 36.67 % (P<0.05). But there was no significant difference in the changes of abnormal rate of serum HA content in IFN-g group. Serum IV-C content decreased remarkably in SA-B group after treatment (P<0.05), but it increased in IFN-g group. There was no significant difference in serum LM and P-III-P contents before and after treatment in both groups. Overall, the first three markers of 9 patients in SA-B group were lowered by ≥30 % after treatment, but of only one patient in IFN-g group (Tables 4-6).

Changes of serum liver function
There was no difference in liver function (Alb, globulin, Serum ALT, AST and GGT activities, serum total and direct bilirubin) between the two groups before treatment. Alb was within normal ranges but globulin was much higher than the normal before treatment in both groups. Alb increased significantly after treatment in IFN-g group (P<0.05), but had no change in SA-B group. There was no significant change of globulin before and after treatment in both groups. The abnormally high ALT activities before the treatment in SA-B group decreased significantly in the first month (P<0.05) and maintained at low levels in the following months of the treatment. In terms of individual subject, ALT activities of 14 patients (46.67 %) in IFN-g group and 7 patients (23.33 %) in SA-B group continued to increase after treatment, but with no significant difference between the two groups (P<0.05), (Table 7). AST activities decreased after treatment in both groups, but without significant difference (Table 8).
      GGT activities decreased after treatment in both groups, without significant difference. Serum total and direct bilirubin content in both groups were close to the upper limit of normal ranges. Total bilirubin contents decreased after treatment in both groups without significant difference. Direct bilirubin contents decreased significantly after treatment in both groups. The remarkable decrease of direct bilirubin in SA-B group started in the 3rd treatment.

Changes of liver ultrasound imaging
There was no obvious change of portal veins and splenic veins before and after treatment in the two groups. SA-B showed an improving tendency based on the score of ultrasound imaging. The scores of only two patients in SA-B group rose (deterioration). But scores of 11 patients increased in the control group. There was significant difference between the two groups
(P<0.05), (Table 9).

Table 4  Serum HA,LM,IV-C and PⅢP contents before, during and after treatment

^aP<0.05, ^bP<0.01, vs before treatment; ^cP<0.05, vs during treatment.

Table 5  Abnormal rates of serum fibrotic markers before and after treatment (abnormal rate(%) = number of abnormal cases/total cases)

^aP<0.05, vs before treatment.

Table 6  Decrease of 4 serum fibrotic markers >30 % after treatment in two groups

^aP<0.05, vs IFN-g

Table 7  Change of ALT activities before and after treatment n (%)

^aP<0.05, vs IFN-g.

Table 8  Change of AST activities before and after treatment n (%)

Table 9  Change of ultrasound image scoring

Comparison of x² test between two groups, ^aP<0.05

Changes of serum viral markers
HBsAg of each patient in both groups was positive before treatment. No changes happened for HBsAg/anti-HBs after treatment. HBV-DNA of 4 patients in each group became negative after treatment. HBeAg of 13 patients in IFN-g group and of 15 patients in SA-B group were positive before treatment. But after treatment, 4 patients in IFN-g group and 3 patients in SA-B group became negative. And at the same time, 2 patients in each group were positive in anti-HBe.

Changes of symptoms and signs
After 6 months treatment, major symptoms and signs such as anorexia, general fatigue, weakness and soreness in lumbar regions and knees, insomnia, yellow urine and hypochondriac pain were remarkably relieved in both groups.

Side effects and safety
The results of EKG, renal function, regular blood test and regular urine test showed no change during the treatment in SA-B group. There were no side effects or negative responses in SA-B group. Fifteen patients in IFN-g group had fever at the beginning of the treatment. One patient was unable to tolerate the side effect, thus terminating the trial. The other patients succeeded in finishing the clinical trial after the disappearance of fever. One patient in IFN-g group had transient decrease of leukocytes. Under careful observation, the patient recovered after treatment (without stopping drug administration or using special treatment). No other side effect was noticed.

Analysis of factors affecting anti-fibrotic treatment effect
Histopathological fibrotic stages before and after treatment were the evidence for evaluating the drug efficacy. It was divided into improved, stable and deteriorated groups. The analysis of liver function and serum fibrotic markers before treatment showed that the effect of SA-B in anti-fibrosis was related to the level of liver injury before treatment. The patient having regression of fibrosis after treatment had much lower ALT and AST activities and total bilirubin content before treatment than those having aggravation of fibrosis after treatment. There was no obvious correlation among the above factors in IFN-g group (Tables 10 and 11).

Table 10  Correlation between change of fibrotic stages before and after treatment,liver function and serum fibrotic markers before treatment in SA-B group

Compared with elevation of stages,^aP<0.05, ^bP<0.01

Table 11  Correlation between change of fibrotic stages before and after treatment, liver function and serum fibrotic markers before treatment in IFN-g group

DISCUSSION
The hepatic fibrosis is the important pathological feature of chronic liver diseases, which is characterized by HSC activation and the overproduction of extracellular matrix^[20-36]. Therefore, inhibition or reversion of hepatic fibrosis is one of the main therapeutic strategies^[37-50]. In this study, we found the SA-B effect on liver fibrosis in hepatitis B for the first time, through the double blind trial with IFN-g as control drug and the observation of liver histopathology, serum fibrotic markers, liver function, liver ultrasound imaging, and symptoms and signs before and after treatment.

SA-B could effectively reverse fibrosis in hepatitis B
The 10^th International Congress of Gastroenterology, Los Angeles, September 1994 suggested the etiological base in terms of the diagnosis of chronic hepatitis, making inflammatory grading criteria according to the severity of histological inflammatory necrosis and fibrotic stage standards according to the level of liver fibrosis. Currently liver biopsy is still the "Gold Standard"for scoring fibrosis. But the great difference in the same liver specimens limited the value of biopsy, making it difficult to evaluate the fibrosis generally and correctly. Liver ultrasound imaging could reflect the specific images of fibrosis. Semi-quantitative evaluation of liver fibrosis and cirrhosis was simple and easily accessible. It was beneficial in the general evaluation of fibrosis, but it failed to precisely reflect the anti-fibrotic effect of the drug because of the insufficiency of quantitative determination. At the same time, multiple factors would affect serum fibrotic markers in the evaluation. It was favorable to combine multiple methods in evaluation. Total 120 biopsy specimens before and after treatment showed that serum fibrotic markers generally rose with the aggravation of fibrotic stages and inflammatory degrees. But the dispersion degree was large. The correlation was better in HA, IV-C and P-III-P. And LM was generally within the normal ranges.
      Adapting to the current situation in diagnosis of liver fibrosis, histological fibrotic stages before and after treatment were fundamental in the evaluation of the efficacy in this trial. Improvement rates of fibrotic stages and inflammatory grades were 36.67 %(11/30) and 40 %(12/30) in SA-B group, and were 30 %(9/30) and 36.67 %(11/30) in IFN-g group, with no significant differences between the two groups. Fibrotic stages of 4 patients decreased more than 2 stages in SA-B group, but none in IFN-g group. HA and IV-C contents decreased significantly after treatment in SA-B group (P<0.05). The abnormal rate of HA was lowered from 80 % to 36.67 % in SA-B group (P<0.05). In the overall analysis of 4 serum fibrotic marker, 9 patients (30 %) in SA-B group had simultaneous decrease of 3 markers, better than one patient in IFN-g group. Scores of liver ultrasound imaging with 2 patients in elevation in SA-B group was better than in IFN-g group with 11 patients in elevation, with significant difference. General analysis of liver biopsy, serum fibrotic markers and ultrasound imaging showed superior effect of SA-B to IFN-g.
      There was no significant change in HBV antigen and antibody systems after treatment in both groups. Anti-fibrotic effect of SA-B or IFN-g was not related to anti-viral effect. Both drugs were favorable in the relief of symptoms and signs.

Factors affecting anti-fibrotic effect of SA-B
After categorizing the changes of histological fibrotic stages, we analyzed the factors affecting anti-fibrotic effect of SA-B through the analysis of serum fibrotic markers and liver functions before treatment. Before treatment, ALT and AST activities and total bilirubin contents in fibrosis improvement group were significantly lower than those in fibrosis aggravation group. The effect was not related to the contents of serum fibrotic markers before treatment. Severity of liver inflammatory injury was the major factor affecting the anti-fibrotic effect of SA-B. SA-B had a satisfactory effect in regression of fibrosis with minor liver injury prior to treatment, but had no favorable results for severe liver injury prior to treatment. The fibrotic stage was not the major factor affecting the anti-fibrotic effect. There was no relationship in IFN-g group. The result was instructive in the clinical application of SA-B. It was good anti-fibrotic effect in the treatment of fibrosis patients with minor inflammatory injury.

No side effects or toxicity of SA-B
There was no obvious negative response in SA-B group during the whole treatment. It showed high security in the 6-month administration. Fever and transient decrease of leukocytes were noticed in IFN-g group.The rate of fever occurrence was 50 %, but it did not affect the completion of the whole treatment.

REFERENCES
1    Xu DS. Dan Shen (Radix Salviae Miltorrhizae): Biology and Application. Beijing Science Press,1990
2    Wu YS, Yang CH, Jiang JF. Primary report of radix salviae miltorrhizae in treatment of late-stage schistosomial cirrhosis
      and splenomegaly. Zhonghua Yixue Zazhi 1958:542-545
3    Yu YX, Yang HQ, Zhu LZ, Cao L, Liu ZL, Zhu LR, Zhang EK, Tian YM, Shen HC, Teng SC. Clinical trial of large-dose radix salviae
      miltorrhizae in treatment of liver fibrosis. Shanghai Zhongyiyao Zazhi 1994: 8-10
4    Chen ZX, Gu WH, Huang HZ, Yang XM, Sun CJ, Chen WZ, Dong YL, Ma HL. Research of water soluble salvianolic acids in radix
      salviae miltorrhizae. Yaoli Tongbao 1981; 16: 24-25
5    Liu P, Mizoguji Y, Morizawa S. Anti-fibrotic effect of magnesium- salvianolic acid B on liver fibrosis rat. Zhongguo Yiyao Xuebao
      1993; 8: 65-67
6    Hu YY, Liu P, Liu C, Gu HT, Liu CH, Ji G. Influence of extracts from radix salviae miltorrhizae on CCl4 and DMN induced liver
      fibrosis rat. Shanghai Zhongyiyao Zazhi 1999:7-10
7    Liu P, Liu NM, Xu LM, Hu YY, Ji G, Liu C. Effect of salvianolic acid B in direct protection of liver cells of original generation rat
      damaged by CCl4 in vitro. Zhongguo Zhongyao Zazhi 1997; 22: 303-305
8    Liu P, Mizoguzhi Y, Morisawa S. Effect of magnesium lithossperamate B on D-galactosamine induced liver injury rat. Zhongguo
      Zhongxiyi Jiehe Zazhi 1995; 1: 291-293
9    Xu LM, Liu C, Liu P. Effect of salvianolic acid B on regeneration, form and extra-cellular matrix synthesis of liver lipocytes of
      second generation rat. Zhonghua Ganzangbing Zazhi 1996; 4: 86-89
10  Jiang SL, Yan XX, Shun YF. The treatment of hepatic fibrosis. Shijie Huaren Xiaohua Zazhi 2000; 8: 684-686
11  Rockey DC, Chung JJ. Interferon gamma inhibits lipocyte activation and extracellular matrix mRNA expression during
      experimental liver injury: implications for treatment of hepatic fibrosis. J Investig Med 1994; 42: 660-70
12  Pancre V, Wolowczuk I, Guerret S, Copin MC, Delanoye A, Capron A, Auriault C. Protective effect of rSm28GST-specific
      T cells in schistosomiasis: role of gamma interferon. Infect Immun 1994; 62: 3723-3730
13  Grossman HJ,White D,Grossman VL,Bhathal PS.Effect of interferon gamma on intrahepatic haemodynamics of the cirrhotic rat
      liver. J Gastroenterol Hepatol 1998;13:1058-1060
14  Oide H, Watanabe S, Sato N. The effect of interferon gamma on hepatic haemodynamics.
      J Gastroenterol Hepatol 1998; 13: 975-976
15  Hoffmann KF, Caspar P, Cheever AW, Wynn TA. IFN-gamma, IL-12, and TNF-alpha are required to maintain reduced liver
      pathology in mice vaccinated with Schistosoma mansoni eggs and IL-12. J Immunol 1998;161:4201-4210
16  Bai WY, Yan XX, Feng LY. Recent progression of hepatic fibrosis research. Shijie Huaren Xiaohua Zazhi 2000; 8: 1267-1268
17  Xu YQ, Wang BE, Cao HG. Ultrasound imaging diagnosis of liver fibrosis.Zhonghua Ganzangbing Zazhi 1998;6:245
18  Proposal of prevention and treatment of viral hepatitis (temporary). Zhonghua Neike Zazhi 1995; 34: 788-791
19  Wang TL, Zhang TH. Development of pathological category of viral hepatitis. Zhonghua Binglixue Zazhi 1996; 25: 67-69
20  Jian HQ, Zhang XL. The mechanisms of hepatic fibrosis. Shijie Huaren Xiaohua Zazhi 2000; 8: 687-689
21  Chen WH, Liu P, Xu GF, Lu X, Xiong WG, Li FH, Liu CH. Role of lipid peroxidation in liver fibrogenesis induced by
      dimthylnitrosamine. Shijie Huaren Xiaohua Zazhi 2001; 9: 645-648
22  Liu C, Chen W, Liu P, Wang Z, Hu Y, Liu C. Changes of lipid peroxidation in liver fibrogenesis induced by dimethylnitrosamine
      and drugs?intervention. Zhonghua Ganzangbing Zazhi 2001; 9: 18-20
23  Neubauer K, Saile B, Ramadori G. Liver fibrosis and altered matrix synthesis. Can J Gastroenterol 2001;15:187-193
24  Paradis V, Perlemuter G, Bonvoust F, Dargere D, Parfait B, Vidaud M, Conti M, Huet S, Ba N, Buffet C, Bedossa P. High glucose
      and hyperinsulinemia stimulate connective tissue growth factor expression: a potential mechanism involved in progression to
      fibrosis in nonalcoholic steatohepatitis. Hepatology 2001; 34: 738-744
25  Schneiderhan W, Schmid-Kotsas A, Zhao J, Grunert A, Nussler A, Weidenbach H, Menke A, Schmid RM, Adler G, Bachem MG.
      Oxidized low-density lipoproteins bind to the scavenger receptor, CD36, of hepatic stellate cells and stimulate extracellular
      matrix synthesis. Hepatology 2001; 34: 729-737
26  Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells.
      Semin Liver Dis 2001; 21: 373-384
27  Vaillant B, Chiaramonte MG, Cheever AW, Soloway PD, Wynn TA. Regulation of hepatic fibrosis and extracellular matrix
      genes by the Th response: new insight into the role of tissue inhibitors of matrix metalloproteinases.
      J Immunol 2001;167: 7017-7026
28  Breitkopf K, Lahme B, Tag CG, Gressner AM. Expression and matrix deposition of latent transforming growth factor
      beta binding proteins in normal and fibrotic rat liver and transdifferentiating hepatic stellate cells in culture.
      Hepatology 2001; 33: 387-396
29  Bruck R, Genina O, Aeed H, Alexiev R, Nagler A, Avni Y, Pines M. Halofuginone to prevent and treat thioacetamide-induced
      liver fibrosis in rats. Hepatology 2001; 33: 379-386
30  Watanabe T, Niioka M, Hozawa S, Kameyama K, Hayashi T, Arai M, Ishikawa A, Maruyama K, Okazaki I. Gene expression
      of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by carbon tetrachloride.
      J Hepatol 2000; 33: 224-235
31  Nakamura T, Sakata R, Ueno T, Sata M, Ueno H. Inhibition of transforming growth factor beta prevents progression of liver
      fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats. Hepatology 2000; 32: 247-255
32  Castera L, Hartmann DJ, Chapel F, Guettier C, Mall F, Lons T, Richardet JP, Grimbert S, Morassi O, Beaugrand M, Trinchet JC.
      Serum laminin and type IV collagen are accurate markers of histologically severe alcoholic hepatitis in patients with cirrhosis.
      J Hepatol 2000; 32: 412-418
33  Brenner DA, Waterboer T, Choi SK, Lindquist JN, Stefanovic B,Burchardt E,Yamauchi M,Gillan A,Rippe RA. New aspects
      of hepatic fibrosis. J Hepatol 2000; 32:32-38
34  Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000; 46: 443-446
35  Levy MT, Trojanowska M, Reuben A, Friedman SL. Evaluation of fibrosis and hepatitis C. Am J Med 1999;107:27S-30S
36  Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury.
      J Biol Chem 2000; 275: 2247-2250
37  Angelico M, Di Paolo D, Trinito MO, Petrolati A, Araco A, Zazza S, Lionetti R, Casciani CU, Tisone G. Failure of a reinforced
      triple course of hepatitis B vaccination in patients transplanted for HBV-related cirrhosis. Hepatology 2002; 35: 176-181
38  Lim KN, Casanova RL, Boyer TD, Bruno CJ. Autoimmune hepatitis in African Americans: presenting features and response to
      therapy. Am J Gastroenterol 2001;96: 3390-3394
39  Moreau R, Asselah T, Codat B, De Kerguence C, Pessione F, Bernard B, Poynard T, Binn M, Grange JD, Valla D, Lebrec D.
      Comparison of the effect of terlipressin and albumin on arterial blood volume in patients with cirrhosis and tense treated by
      paracentesis: a randomised polit study. Gut 2002; 50: 90-94
40  Riley TR, Bhatti AM. Preventive strategies in chronic liver disease:part II,cirrhosis. Am Fam Physician 2001;64:1735-1740
41  Rajan S, Liebman HA. Treatment of hepatitis C related thrombocytopenia with interferon alpha.
      Am J Hematol 2001; 68: 202-209
42  Border WA, Noble N. Maximizing hemodynamic-independent effects of angiotensin II antagonists in fibrotic diseases.
      Semin Nephrol 2001; 21: 563-572
43  Freeze HH. Congenital disorders of glycosylation and the pediatric liver. Semin Liver Dis 2001; 21: 501-516
44  Tolan DJ, Davies MH, Millson CE. Fibrosing cholestatic hepatitis after liver transplantation in a patient with hepatitis C and
      HIV infection. N Engl J Med 2001; 345: 1781
45  Simeonova PP, Gallucci RM, Hulderman T, Wilson R, Kommineni C, Rao M, Luster MI. The role of tumor necrosis factor-alpha
      in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl Pharmacol 2001; 177: 112-120
46  Di Martino V, Rufat P, Boyer N, Renard P, Degos F, Martinot-Peignoux M, Matheron S, Le Moing V, Vachon F, Degott C,
      Valla D, Marcellin P. The influence of human immunodeficiency virus coinfection on chronic hepatitis C in injection drug users:
      a long-term retrospective cohort study. Hepatology 2001;34: 1193-1199
47  Bini EJ, Weinshel EH, Generoso R, Salman L, Dahr G, Pena-Sing I, Komorowski T. Impact of gastroenterology consultation on
      the outcomes of patients admitted to the hospital with decompensated cirrhosis. Hepatology 2001; 34: 1089-1095
48  Romagnuolo J, Jhangri GS, Jewell LD, Bain VG. Predicting the liver histology in chronic hepatitis C: how good is the clinician?
      Am J Gastroenterol 2001; 96: 3165-3174
49  Angulo P, Jorgensen RA, Lindor KD. Incomplete response to ursodeoxycholic acid in primary biliary cirrhosis: is a double
      dosage worthwhile? Am J Gastroenterol 2001; 96: 3152-3157
50  Nakamuta M, Ohta S, Tada S, Tsuruta S, Sugimoto R, Kotoh K, Kato M, Nakashima Y, Enjoji M, Nawata H. Dimethyl sulfoxide
      inhibits dimethylnitrosamine-induced hepatic fibrosis in rats. Int J Mol Med 2001; 8: 553-560

Edited by Ma JY